A near-infrared-emitting fluorescent probe for monitoring mitochondrial pH

Article abstract of DOI:10.1039/c4cc01390e

We demonstrate a new small molecule fluorescent probe, possessing near-infrared (NIR) emission and an unusually large Stokes shift. It can be readily taken up by live cells and mitochondria, and track subtle pH changes with effectively reduced biological background fluorescence and improved measurement accuracy.

Full text of DOI:10.1039/c4cc01390e

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A near-infrared-emitting fluorescent probe for
monitoring mitochondrial pH†
Cite this: Chem. Commun., 2014,
50, 7184
Ping Li, Haibin Xiao, Yinfang Cheng, Wen Zhang, Fang Huang, Wei Zhang,
Hui Wang and Bo Tang*
Received 23rd February 2014,
Accepted 9th May 2014
DOI: 10.1039/c4cc01390e
www.rsc.org/chemcomm
We demonstrate a new small molecule fluorescent probe, posses- important to develop ideal fluorescent probes for accurate measure-
sing near-infrared (NIR) emission and an unusually large Stokes ment of subtle changes in pH within mitochondria. These probes
shift. It can be readily taken up by live cells and mitochondria, and should possess the following unique characteristics: (1) maximum
track subtle pH changes with effectively reduced biological back- fluorescence emission wavelengths are in the NIR region for lower
ground fluorescence and improved measurement accuracy.
interference from background fluorescence, (2) a large Stokes shift
for minimizing reabsorption of emitted photons from chromo-
Mitochondria are the double-membrane-bound subcellular phores, as well as good mitochondria-targeted capability. Although
organelles providing enormous metabolic functions,1–4 such as protein probes15 and small molecule probes16 have been reported
producing much of the cellular energy, regulating the cellular for mitochondrial pH detection, NIR fluorescent probes with a
redox state, generating most of the cellular reactive oxygen species large Stokes shift and good targeting capability are rare.
(ROS),5–7 and initiating cellular apoptosis.8,9 The specific pH values
To solve these problems, we have fabricated a novel fluorescent
of mitochondria influence directly all biochemical processes. So probe, (E)-3-((E)-2-chloro-3-((E)-2-(1-ethyl-3,3-dimethylindolin-2-
this parameter has to be severely controlled within a narrow range, ylidene)ethylidene)cyclohex-1-enyl)-1-(4-(dimethylamino)phenyl)-
and aberrations of the normal pH homeostasis may lead to prop-2-en-1-one (Spring Red), based on a long p-conjugated
mitochondrial dysfunction. Accurate and sensitive measurements system containing a dimethylamino group (Scheme 1). The
of mitochondrial pH (pHmit) dynamics in living cells are therefore relatively long conjugated system with this structure is expected
crucial to the understanding of mitochondrial biology. However, to fluoresce in the NIR region. Moreover, from existing studies,
despite previous extensive investigations focussing on mitochon- we consider that structural asymmetry of Spring Red may
drial biology, a great variety of their fundamental cellular functions account for a large Stokes shift, ascribing to more energetically
still remain elusive, which is hampered severely by the shortage of distinct ground and excited states.17
ideal approaches for tracking mitochondrial biological events,
including pH changes.
For the purpose of reporting alterations of mitochondrial pH, a
pH-sensitive dimethylamino group is introduced to tune fluores-
It is now generally accepted that organic fluorescent probes are cent properties of the scaffold. Under relatively acidic conditions,
some of the cornerstones of modern bioanalysis and real-time lone pair electrons of nitrogen conjugating with the p-electron
bioimaging technologies.10–12 They offer excellent sensitivity and system disappeared upon protonation. As a consequence, p–p
high spatial resolution while minimally perturbing the live cells conjugation fading results in gradual fluorescence decrease and
and organisms. In particular, near-infrared (NIR) emission fluores- blue shift of emission wavelength. In contrast to this, the fluor-
cent probes are able to dramatically reduce influence of biological escent intensity of the probe should increase with the rise in pH.
background fluorescence, which has grown into an important tool We confirmed that the protonation of the dimethylamino group
in widespread exploration of biological processes in live cells and could control fluorescence properties of Spring Red by molecular-
in vivo.13,14 Because small changes in pHmit may result in a signi- orbital (DFT) calculations (Fig. S1, ESI†). Moreover, a resonance
ficant impact on mitochondrial biological functions, it is very
College of Chemistry, Chemical Engineering and Materials Science, Collaborative
Innovation Center of Functionalized Probes for Chemical Imaging, Key Laboratory
of Molecular and Nano Probes, Ministry of Education, Shandong Normal University,
Jinan 250014, P. R. China. E-mail: tangb@sdnu.edu.cn; Tel: +86 531-8618-0017
† Electronic supplementary information (ESI) available: Detailed synthetic pro-
cedure and characterization data. See DOI: 10.1039/c4cc01390e
Scheme 1 Proposed mechanism for fluorescence change of Spring Red.
7184 | Chem. Commun., 2014, 50, 7184--7187
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structure of Spring Red with a lipophilic cation (Scheme S2,
ESI†) is apt to accumulate in mitochondria due to the mito-
chondrial membrane potential.18 Thus, this well-designed com-
pound is surmised to be capable of labelling alkali organelles,
mitochondria, and reversibly indicating the changes in mito-
chondrial pH. This compound was easily prepared by facile
synthesis, and characterized by UV/Vis, 1H NMR, 13C NMR and
HRESI-MS spectroscopy (see ESI†).
The spectral properties of Spring Red were studied system-
atically. The UV/Vis absorption or excitation and emission
profiles are evidenced in ESI† (Fig. S2). As expected, its emis-
sion wavelength is in the NIR region, and its Stokes shift is
150 nm. As a result, the accuracy of mitochondrial pH measure-
ment is drastically improved, which provides an ideal high-
resolution fluorescent imaging probe for organelle labelling.
Fluorescent responses of Spring Red toward various pH values
were examined systematically in universal buffer solution (0.1 M
citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M
KCl),19 in order to test the hypothesis concerning regulatory
function of the dimethylamino group for fluorescence properties.
As shown in Fig. 1, the pH titration results imply that Spring Red
Fig. 2 Confocal fluorescence imaging of Spring Red in HepG2 cells (A)
and zebrafish (B). (a) Spring Red (30 mM, lex = 543 nm, collected in the range
of 650–800 nm). (b) Mito-Tracker Green (25 nM, lex = 488 nm, collected in
the range of 495–520 nm). (c) Merged images of a and b. (d) Spring Red in
zebrafish. (e) Brightfield image. (f) Merged images of d and e.
fluorescence is remarkably pH-dependent from pH 5.0 to 8.0 with Red fluoresced brightly in HepG2 cells (Fig. 2a). From a contrasting
a pKa of 6.3, and the fluorescence intensity is greater in alkali merged image of the probe and Mito-Tracker Green staining, we
than in acidic environments. Particularly, Spring Red gives a found that fluorescence brightness of Spring Red within the cell was
strong NIR emission peaked at 680 nm and pH B 8.0, and very heterogeneous, well overlapped with Mito-Tracker Green
thereby it affords a useful pattern to visualize intracellular imaging with a colocalization coefficient of 0.91 (Fig. 2a–c).
mitochondria due to an alkali environment inside mitochon- Obviously, it gives rise to brighter fluorescence in mitochondria.
dria, and also indicates changes in mitochondrial pH. Further Further evaluation for targeting mitochondria capability of Spring
investigations demonstrate that fluorescence of Spring Red is Red was carried out in zebrafish. As shown in Fig. 2d–f, there is
basically unperturbed by relative biological components includ- more extensive fluorescence in the heart region of zebrafish treated
ing common cations and ROS (Fig. S3, ESI†).
with Spring Red, mainly due to the high concentration of mito-
We next performed colocalization imaging experiments in chondria in heart tissue.20 In addition, the similar results were
human hepatoma cells (HepG2) to observe intracellular distribution obtained from localization-imaging experiments of Mito-Tracker
patterns of Spring Red. Cultured cell lines were stained with Spring Green in zebrafish (Fig. S4, ESI†). Moreover, three analogues of
Red for 10 min, and then we studied uptake and internalization Spring Red were synthesized, and colocalization-imaging experi-
of the probe by live cells and mitochondria. At the same time, ments showed that they also accumulate in mitochondria to some
Mito-Tracker Green, a commercial mitochondrial dye, serves to extent (Fig. S5, ESI†), which indicate that the main structure of
label mitochondria in order to assess targeting mitochondria cap- Spring Red plays an important role in targeting mitochondria.
ability of Spring Red. Direct visual evidence suggested that Spring These results clearly suggest that Spring Red can diffuse fast into
mitochondria and fluoresce strongly because of relatively alkali
conditions in mitochondria, and is well suitable for in vivo imaging.
For studying whether the probe possesses dynamic imaging
features, Spring Red was used to monitor intracellular pH changes.
The in situ calibration curve for pH measurements in HepG2 cells
was obtained using high K+ buffer and ionophore nigericin21 in bath
solutions of defined pH (Fig. 3). The fluorescence intensity of Spring
Red (Fig. 3a) inside cells enhances proportionally with an increase of
pH values. The mean intensity at different pH in the cells was
quantified as in Fig. 3b, showing pH-dependence over the pH range
of 4.0–8.5, with the regression equation Y = 0.3714x À 1.883 (R2 =
0.9958). Moreover, leakage basically did not occur over the course of
imaging experiments (even 30 min) and the probe was resistant to
photobleaching (Fig. S6–S8, ESI†), as well as low cell toxicity (Fig. S9,
ESI†), which permit a prolonged observation time in vivo.
Fig. 1 Fluorescence spectral changes of Spring Red (30 mM) with various
It was reported that lactate and pyruvate were exploited to
cause mitochondria acidification.15a Under these conditions,
pH values from 2.2 to 9.0 (lex = 530 nm, 10 mM universal buffer). Inset:
sigmoidal fitting of the pH-dependent fluorescence intensity at 680 nm.
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Fig. 3 (a) Fluorescent confocal microscopy images of Spring Red (30 mM)
in HepG2 cells defined at pH 4.0–8.5. (b) Intracellular pH calibration curve
of Spring Red in HepG2 cells from (a). DF = F À F5.0 (F: mean fluorescence
intensity at different pH values; F5.0: mean fluorescence intensity at pH = 5.0).
The excitation wavelength was 543 nm and the images were collected at
650–800 nm.
Fig. 5 Representative fluorescence images (pseudo-color) of mice injected
with Spring Red during LPS-mediated inflammatory response in vivo. (a) Only
LPS was injected for control. (b) Only Spring Red was injected. (c) LPS was
injected into the peritoneal cavity of the mice, followed by injection with
Spring Red (50 mM). (d) Quantification of fluorescence emission intensity
from group a–c. Representative images of mice from replicate experiments
(n = 3). The mean fluorescence intensity from the mice peritoneal cavity
was output. The mice were imaged with an excitation filter (530 nm) and
an emission filter (670 nm).
fluorescence images of HepG2 cells were captured. During normal
incubations, HepG2 cells exhibited a mitochondrial pH of
7.96 Æ 0.13 (Fig. 4a). In the presence of glucose, pH did not change
basically from the analysis of images and fluorescent intensity data
output (Fig. 4b). Upon the treatment of lactate and pyruvate, steady
decreases occurred in pH from 7.97 to 7.14. Subsequently, after
lactate and pyruvate were removed, pH values reversed gradually to
some degree from 7.14 to 7.45. The pH did not get back to the
original level after washout in contrast to the previous report,
probably due to slight leakage of the probe in the cells treated with
lactate/pyruvate, which led to 0.51 pH unit difference to the initial
level. However, the tendency of mitochondrial pH changes measured
by this method is in agreement with that under similar conditions in
previous reports.15a In addition, we applied Spring Red in HepG2
cells to determine the pH changes in the apoptosis process, induced
by L-buthionine sulfoximine (BSO).22 The fluorescence intensity of
Spring Red decreased in BSO-treated cells during 1 h, indicating a
decline of the pH value in cellular apoptosis (Fig. S10, ESI†).23 All the
results suggest that this fluorescent probe can real-time monitor
small pH alterations inside mitochondria.
Finally, we investigated the application of Spring Red to visualize
pH changes in an abdominal inflammation model induced by
lipopolysaccharides (LPS).24–26 Two mice suffered an acute inflam-
matory response in the peritoneal cavity via skin-pop injection of LPS
(Fig. 5a and c). One of the mice was then injected with Spring Red
(Fig. 5c). This mouse treated with both LPS and Spring Red (Fig. 5c)
exhibited lower fluorescence intensity (pseudo-color) than that of
only Spring Red-treated mouse (Fig. 5b), indicating the decrease of
the pH value in the inflammatory tissues.27–29 As a control, the mice
treated with LPS but not with Spring Red showed almost no
fluorescence (Fig. 5a). The intensity was quantified, and the data
imply that the fluorescence intensity of Spring Red in the mice with
abdominal inflammation shows an obvious decrease (Fig. 5d).
Taken together, these results demonstrate that Spring Red is useful
for in vivo imaging and can detect the pH changes in small animals.
In conclusion, we have presented a novel near-infrared-emitting
fluorescent probe for sensing subtle pH changes with an unprece-
dented large Stokes shift. It also exhibits good photostability and low
cytotoxicity. Fluorescence images of live cells and in vivo reveal that it
can easily diffuse into mitochondria and reliably report pH changes
under acidic conditions, cellular apoptosis or inflammation
response. Overall, the merits of Spring Red impart a faithful estima-
tion of small mitochondrial pH alterations in complicated biological
systems, and also it has been the first NIR fluorescent emission
probe for measurement of mitochondrial pH up to now. Moreover,
the proposed method provides a new strategy for quantitative
biology of molecular events inside other subcellular organelles.
This work was supported by the 973 Program (2013CB933800),
the National Natural Science Foundation of China (21227005,
21390411, 91313302, 21035003a, 21205073) and the Program for
Changjiang Scholars and Innovative Research Team in University.
References
1 Y. Yamada and H. Harashima, Adv. Drug Delivery Rev., 2008, 60,
1439–1462.
2 R. Rizzuto, D. De Stefani, A. Raffaello and C. Mammucari, Nat. Rev.
Mol. Cell Biol., 2012, 13, 566–578.
3 D. C. Wallace, Nat. Rev. Cancer, 2012, 12, 685–698.
4 S. Fulda, L. Galluzzi and G. Kroemer, Nat. Rev. Drug Discovery, 2010,
9, 447–464.
Fig. 4 (a) Confocal microscopy image of Spring Red (30 mM) in HepG2
cells under different conditions. (b) Effect of glucose and lactate/pyruvate
perfusion on mitochondrial at different time. The excitation wavelength
was 543 nm and the images were collected at 650–800 nm.
7186 | Chem. Commun., 2014, 50, 7184--7187
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5 B. Chance, H. Sies and A. Boveris, Physiol. Rev., 1979, 59, 527–605.
6 R. S. Balaban, S. Nemoto and T. Finkel, Cell, 2005, 120, 483–495.
17 J. F. Araneda, W. E. Piers, B. Heyne, M. Parvez and R. McDonaid,
Angew. Chem., Int. Ed., 2011, 50, 12214–12217.
7 A. M. James, Y. Collins, A. Logan and M. P. Murphy, Trends Endocrinol. 18 (a) L. V. Johnson, M. L. Walsh, B. J. Bockus and L. B. Chen, J. Cell
Metab., 2012, 23, 429–434.
8 V. Gogvadze, S. Orrenius and B. Zhivotovsky, Apoptosis, 2009, 14,
624–640.
Biol., 1981, 88, 526–535; (b) S. T. Smiley, M. Reers, C. Mottola-
Hartshorn, M. Lin, A. Chen, T. W. Smith, G. D. Steele and L. B.
Chen, Proc. Natl. Acad. Sci. U. S. A., 1991, 88, 3671–3675.
9 V. Gogvadze, S. Orrenius and B. Zhivotovsky, Semin. Cancer Biol., 19 H. M. Kim, M. J. An, J. H. Hong, B. H. Jeong, O. Kwon, J. Y. Hyon,
2009, 19, 57–66.
10 T. Ueno and T. Nagano, Nat. Methods, 2011, 8, 642–645.
S. C. Hong, K. J. Lee and B. R. Cho, Angew. Chem., 2008, 120,
2263–2266.
´
´
11 M. Fernandez-Suarez and A. Y. Ting, Nat. Rev. Mol. Cell Biol., 2008, 9, 20 A. T. Hoye, J. E. Davoren, P. Wipf, M. P. Fink and V. E. Kagan,
929–943. Acc. Chem. Res., 2008, 41, 87–97.
12 S. Chen, Y. Hong, Y. Liu, J. Liu, C. W. T. Leung, M. Li, R. T. K. Kwok, 21 M. H. Lee, J. H. Han, J. H. Lee, N. Park, R. Kumar, C. Kang and
E. Zhao, J. W. Y. Lam, Y. Yu and B. Tang, J. Am. Chem. Soc., 2013,
135, 4926–4929.
13 J. V. Frangioni, Curr. Opin. Chem. Biol., 2003, 7, 626–634.
14 S. A. Hilderbrand and R. Weissleder, Curr. Opin. Chem. Biol., 2010,
14, 71–79.
J. S. Kim, Angew. Chem., Int. Ed., 2013, 52, 6206–6209.
22 J. S. Armstrong, K. K. Steinauer, B. Hornung, J. M. Irish, P. Lecane,
G. M. Birrell, D. M. Peehl and S. J. Knox, Cell Death Differ., 2002, 9,
252–263.
23 D. Lagadic-Gossmann, L. Huc and V. Lecureur, Cell Death Differ.,
2004, 11, 953–961.
15 (a) J. Llopis, J. M. Mccaffery, A. Miyawaki, M. G. Farquhar and
R. Y. Tsien, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 6803–6808; 24 J. E. Parrillo, N. Engl. J. Med., 1993, 328, 1471–1477.
(b) L. Wei-LaPierre, G. Gong, B. J. Gerstner, S. Ducreux, D. I. Yule, 25 C. R. H. Raetz and C. Whitfield, Annu. Rev. Biochem., 2002, 71,
S. Pouvreau, X. Wang, S. S. Sheu, H. Cheng, R. T. Dirksen and
W. Wang, J. Biol. Chem., 2013, 288, 10567–10577; (c) M. Tantama, 26 M. S. Trent, C. M. Stead, A. X. Tran and J. A. Hankins, J. Endotoxin
Y. P. Hung and G. Yellen, J. Am. Chem. Soc., 2011, 133, 10034–10037. Res., 2006, 12, 205–223.
635–700.
16 (a) A. Takahashi, Y. Zhang, V. E. Centonze and B. Herman, 27 A. Lardner, J. Leukocyte Biol., 2001, 69, 522–530.
Biotechniques, 2001, 30, 804–815; (b) D. W. Jung, M. H. Davis and 28 A. Punnia-Moorthy, J. Oral Pathol. Med., 1987, 16, 36–44.
G. P. Brierley, Anal. Biochem., 1989, 178, 348–354.
29 F. Okajima, Cell. Signalling, 2013, 25, 2263–2271.
This journal is ©The Royal Society of Chemistry 2014
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